Protease inhibitor assay

ABSTRACT

Heterogenous and homogenous assays are provided for the detection of protease inhibitory activity in a sample or target compound, taking advantage of the chemiluminescent characteristics of 1,2-dioxetanes. In the heterogenous assay, a peptide bearing a cleavage site for the protease of interest is provided with a first member of a first ligand binding pair at one end, and a first member of a second ligand binding pair at the other end. The other member of the first ligand binding pair is attached to a surface, which binds the peptide, or protease substrate, to the surface. The peptide substrate is combined with the protease and target compound or sample. Substrate cleavage, if not inhibited, is allowed to occur, and any unbound cleaved fragments are removed. An enzyme complexed with the second member of the second ligand binding pair is added, and allowed to bind to any of the (uncleaved) first member of the second ligand binding pair remaining. Unbound complex is removed, and a 1,2-dioxetane substrate for the enzyme is added. If any peptide substrate has not been cleaved, the dioxetane will chemiluminesce, indicating inhibitory activity. In a homogenous assay, the same substrate bears at one end a fluorescent energy accepting moiety, and at the other end a 1,2-dioxetane or precursor. If the substrate is cleaved by the protease, the dioxetane and the fluorescent moiety are not in close physical relationship, and no energy transfer occurrs when the dioxetane is caused to decompose. If cleavage has not occurred, indicating inhibition, when the dioxetane is caused to decompose, energy is transferred to the fluorescing entity, which releases light of a wavelength recognizably distinct from that of the dioxetane.

[0001] This application is a regular National application claimingpriority from Provisional Application, U.S. Application Ser. No.60/038,940 filed Mar. 7, 1997.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention pertains to the use of chemiluminescent1,2-dioxetanes in homogenous or heterogeneous assays to detect proteasesinhibitors.

[0004] 2. Discussion of the Background

[0005] The identification of novel therapeutics that block or inhibitinimical proteases, or proteases that mediate disease conditions, suchas the 11-kd protease encoded by the human immunodeficiency virus1(HIV-1) is a key step in slowing the disease process of AIDS.Retroviral proteases are essential in the process of viral gag-polpolyproteins of the HIV-1 and HIV-2 viruses. There are a few highlyconserved consensus sequences in retroviral polyproteins, one of whichconsists of a pentapeptide (Ser/Thr)-X-X′-(Tyr/Phe)-Pro. Cleavage occursbetween the Tyr or Phe and Pro residues. Blocking activity of theseproteases will interfere with the progression of HIV infection. Althoughpotent drugs which block HIV protease activity have been found, there isan ongoing need to find and develop novel inhibitors.

[0006] Current methods utilized in rapid screening of proteaseinhibitors are subject to many interferences from a variety of sources.The most common non-isotopic approach is a fluorescent assay. In onecase, such as in the detection of HIV protease, the fluorescentsubstrate is labeled with a fluorescent dansyl group on one end of apeptide and a quencher on the other. An increase in fluorescence signaloccurs upon cleavage of the protease due to the fact that the emitterand the quencher are separated as described in Matayoshi et al. 1990,Science 247: 954-958. Fluorescent substrates for other proteases can bedesigned 5 with a terminal fluorophore which emits a fluorescent signalupon cleavage by the enzyme.

[0007] Both of the above assay approaches are commonly used as highthroughput assays for screening large chemical, natural product andcombinatorial libraries. These assays tend to have problems related toautofluorescence of biological components due to the nature of themolecules which are screened. Many of the compounds and natural product10 extracts are colored or fluorescent and are present in the solutionwhen the assay signal is monitored. This results in an assayinterference which limits the detection sensitivity and the dynamicrange of the assay. This interference can easily be interpreted as aninhibition of the enzyme, making it difficult to determine true positiveinhibition, thus, requiring extensive follow-up assays to distinguishtrue positives from the false positives.

[0008] U.S. Pat. No. 5,591,591, assigned to Tropix, describes assays forthe detection of proteases wherein a dioxetane compound bearing aproteolytic enzyme-specific amino acid or peptide, is added to a samplesuspected of containing the protease, and the amino acid is removed byenzymatic reaction, causing the dioxetane to decompose andchemiluminescence.

SUMMARY OF THE INVENTION

[0009] An alternative approach to the above homogeneous approaches is touse homogenous or heterogeneous assays which are not subject tointerference. The present inventors have developed highly sensitiveassays using a chemiluminescent 1,2-dioxetane substrate for highthroughput screening of HIV-1 protease activity. The present inventionprovides the advantage of an assay that it is not subject tointerferences from colored or fluorescent compounds, and therefore ismore sensitive and exhibits a greater dynamic range compared to adirect, fluorescent enzyme assay. This assay, adapted for the detectionof HIV protease inhibitors, utilizes a synthetic peptide substrate I(Fam-spacer-Ser-Gln-Asn-Try-Pro-lle-Val-Gln-spacer-(Biotin)-NH2) whosesequence is derived from the native cleavage site of the Gagpolyprotein. (Fam is used herein to indicate fluorescein). Many highthroughput HIV screening methodologies exist which utilize largequantities of reagents and involve more laborious manipulations. Theassay of the present invention may be advantageously formatted as asimple single plate endpoint assay which is sensitive down to fmoles ofcaptured peptide which is particularly useful for high throughoutscreening, although it may also be presented as a conventional two-steptransfer and dilation capture assay. The assay uses a chemiluminescent1,2-dioxetane alkaline phosphatase substrate in an immunoassay formatfor the sensitive detection of cleaved peptide. The uncleaved peptide isrecognized by an anti-fluorescein alkaline phosphatase conjugatedantibody. Capture conditions have been optimized to assure a linearresponse to cleaved peptide concentration. This response correlates wellto HPLC analysis of cleaved product. The HIV assay has been validatedfor acetyl-pepstatin, a known HIV-1 inhibitor. This robust assay isamenable to automation and can be used to screen large numbers ofcompounds in a cost effective 96 well format.

[0010] In an alternative embodiment, a homogenous assay less sensitiveto color or fluorescence interference than prior art assays is used. Inthis assay, the same peptide is employed, but labeled at one terminuswith fluorescein or other fluorescing energy acceptor, and at the otherend by a 1,2-dioxetane label covalently attached to the peptideterminus. After admixture of the target compound, the dioxetane iscaused to decompose by addition of a chemical (enzymatic ornon-enzymatic) or another trigger, such as application of heat or changein pH. If the peptide has not been cleaved, the dioxetane is in closephysical relationship with the fluorescent energy acceptor, and upontriggering, energy transfer assisted fluorescence is observed. The lighthas a characteristic color dependent on the fluorescent emitter, such asgreen for fluorescein. If the peptide has been cleaved, thechemiluminescent light of the dioxetane itself, typically a bluer light,is observed.

DETAILED DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1. Illustration of two labeled HIV-1 substrates I and IIwhose sequences are derived from a natural processing site for aspartylHIV-1 protease. Incubation of these two HIV-1 peptides with rec HIV-1protease results in a specific cleavage between the Try-Pro bond asreported in Kohl, NE et al.(1988), Proc. Acad. Sci. USA, 85, 4684 andBillich, SW, et al. (1988), J. Biol. Chem. 263, 17905. These arecompared against the 15 prior art substrate as reported in Mayatoshi etal.

[0012]FIG. 2. HIV-1 Protease Assay. Steps include 1. Addition of recHIV-1 protease, acetyl-pepstatin(3) and/or screening compound toNeutrAvidin coated plates, with a preincubation. 2. Addition of HIV-1protease peptide I followed with a 60 min. Incubation at 37° C. 3. Washout cleaved peptide fragment. 4. Add anti-fluorescein alkalinephosphatase antibody to detect uncleaved bound peptide and then 5. AddCSPD/Sapphire II and measure emitted light in a TR717luminometer(Tropix).

[0013]FIG. 3. The phosphate group on the CSPD substrate is cleaved byalkaline phosphatase to generate the unstable anion intermediate 1.Intermediate 1, then undergoes decomposition producing a long-livedemission at 470 nm.

[0014]FIG. 4. HIV-1 protease peptide I titration on Neutravidin coatedplates. 100 ul of diluted peptide was added to various densities ofcoated Neutravidin wells. Plates were washed and peptide was detectedwith an anti-fluorescein alkaline phosphatase conjugate followed withthe addition of CSPD/Sapphire II. Light emission was measured in a TR717microplate luminometer(Tropix).

[0015] FIGS. 5(A&B). Comparison of HIV-1 peptide I cleavage by rec HIV-1protease by HPLC analysis and capture on Neutravidin coated plates. 35nM of rec HIV-1 protease was added to 10 uM HIV peptide in eppendorftubes and incubated at 37° C. At specific time points 0.1% TFA was addedto stop the reaction and tubes placed on ice. Reaction products wereanalyzed by HPLC. Injection volumes were 100 ul and absorbency wasmonitored @490 nm. The percentage of cleaved product was calculated fromthe peak areas. For peptide capture experiments, 100 ul of a 1:50dilution was added to Neutravidin (Sug/ml) coated plates and incubatedat ambient temp. for 60 min. after a wash step. captured peptide wasdetected with anti-fluorescein conjugated alkaline phosphatase antibodyfollowed by another wash step and then the addition of CSPD/Sapphire II.Light emission was measured in a TR717 microplate luminometer(Tropix).

[0016]FIG. 6. Hydrolysis of EDANS/DABCYL HIV-1 protease Prior ArtSubstrate by rec HIV-1 protease. Fluorogenic HIV-1 peptide II was addedto protease buffer pH 4.7 in 96 well plates. Rec HIV-1 protease wasadded at ambient temperature and immediately read over a 105 minute timeperiod on a LS50B fluorescence spectrophotometer (Perkin-Elmer).Excitation was set at 340 nm and emission was set at 490 nm.

[0017]FIG. 7. Hydrolysis and capture of HIV-1 protease substrate I onNeutravidin coated plates. Dilutions of rec HIV-1 protease diluted inprotease buffer pH 5.5 were added to wells previously coated with 0.1ug/ml Neutravidin. 10 pmoles of protease substrate was added to startthe reaction and the plate was incubated for 1 hour at 37° C. After awash step, captured peptide was detected with anti-fluoresceinconjugated alkaline phosphatase antibody followed by another wash stepand then the addition of CSPD/Sapphire II. Light emission was measuredin a TR717 microplate luminometer(Tropix). 100% cleavage of thesubstrate is not obtained for substrate 1. The date has been adjusted toreflect this phenomenon. In actuality, the detected background may bedifferent.

[0018]FIG. 8. Rec HIV-1 protease inhibition by acetyl-pepstatin in aHIV-1 capture assay. 2.5 pmoles of rec HIV-1 protease diluted inprotease buffer pH 5.5 was added to Neutravidin (0.1 ug/ml) coatedwells. Dilutions of acetyl-pepstatin in DMSO were added to wellsfollowed with a preincubation of 5 minutes. 20 pmoles of HIV-1 proteasesubstrate was added and the plate incubated for 1 hour at 37° C. After awash step captured peptide was detected with an anti-fluoresceinconjugated alkaline phosphatase antibody. After a second wash step,CSPD/Sapphire II was added and light emission was measured in a TR717microplate luminometer(Tropix).

[0019]FIG. 9. Screening compound plate-1001 at 25 uM (5% DMSO) for recHIV-1 protease inhibition. 2.5 pmoles of rec HIV-1 protease diluted inprotease buffer pH 5.5 was added to Neutravidin coated plates. 5 ul froma compound screening plate largely composed of drug standards was addedand preincubated for 5 minutes. 20 pmoles of HIV-1 protease substrate Iwas added and the plate was incubated for 60 minutes at 37° C. After awash step, captured peptide was detected with anti-fluoresceinconjugated alkaline phosphatase antibody. After a second wash step,CSPD/Sapphire II was added and light emission was measured in a TR717microplate luminometer(Tropix).

[0020]FIG. 10. HIV FLAG Peptide titration on Neutravidin and Anti-FLAGMI monoclonal antibody coated plates. 100 ul of diluted HIV FLAG peptidewas added to various densities of pre-coated Neutravidin or Anti FLAG MImonoclonal antibody coated plates. Coating was achieved by diluting agiven amount of Neutravidin or Anti-FLAG MI monoclonal antibody and BSAin PBS so as to achieve a total protein concentration of 5 μg/ml. Plateswere incubated for 1 hour at 37° C. followed by a 3× wash with TBS/0.05%Tween/3 mM CaCl₂. 100 ul of a 1:20,000 dilution of Avidix-AP was addedand incubated for 1 hour at room temp. After 3 washes in TBS/Tween/CaCl₂buffer and 1 wash with 1× Tris/1 mM MgCl/3 mMCaCl pH 9.8, 100 ul readyto sue CSPD/Sapphire II was added and wells incubated for 30 min at roomtemp. Luminescence was then measured in a TR717 microplate luminometer(Tropix). The present inventors have discovered novel peptide captureconditions which permit the assay to be performed as a single well(plate) assay as opposed to a 2 step transfer and dilution assay. Asshown in FIG. 12, the signals are relatively the same when done as a onestep or two step assay suggesting that non-specific cleavage of otherassay components by the rec HIV-1 protease is nonexistent.

[0021]FIG. 11. Capture of HIV FLAG and HIV FAM Peptides on Neutravidin(5 ug/ml) coated plates post hydrolysis with rec HIV-1 protease (50 nM).50 nM rec HIV-1 protease was added to 1 uM HIV FLAG or HIV FAM peptidesin protease buffer (0.1 mM NaAcetate, 1 M NaCl, 1 mM EDTA, 1 mM DTT, 1mg/ml BSA, pH 4.7). Biotin at 0.5 mM and 5 Units of enterokinase wereadded to control tubes. Reactions were done for 1 hour at 37° C. ineppendorf tubes. All tubes were then placed on ice to stop the reaction.An additional 50 nM rec HIV-1 protease was added to 2 of the reactiontubes and incubated further for 30 minutes at 37° C. HIV FLAG peptidecontaining tubes were diluted 1:100 (10 nM final) and HIV FAM peptidetubes were diluted 1:100,000 (0.01 nM final) in protease buffer. 100 ulwas added to Neutravidin coated wells (5 ug/ml) and incubated for 1 hourat room temp. Wells were then washed with 3× PBS/Tween/CaCl buffer. 100ul of 1:5000 dilution of Anti FITC-AP Fab Fragment was added to HIV FAMpeptide containing wells and 100 ul of a precomplexed Anti-FLAG MIantibody (1:1000) and Goat anti mouse-AP conjugate (1:10,000) was addedto HIV FLAG peptide containing wells. Plates were incubated foradditional hour at room temp then washed 3× in TBS/Tween/CaCl and 1×Tris/MgCl/CaCl pH 9.8. 100 ul ready to use CSPD/Sapphire II was added towells and incubated 30 minutes at room temp. Luminescence was thenmeasured in a TR717 microplate luminometer.

[0022]FIG. 12. Titration of rec HIV-1 Protease I with 100 nM HIV FLAGPeptide. Diluted rec HIV-1 protease was added to 100 nM HIV FLAG peptidein protease buffer. Reactions were done either in eppendorf tubes ordirectly in wells precoated with Anti-FLAG MI monoclonal antibody (0.1ug/ml). Competing FLAG octapeptide at 2.5 uM was added to control tubesand wells. Incubations were done for 1 hour at 37° C., then eppendorfreaction products transferred to wells, and further incubated for 1 hourat room temp. Plates were washed 3× in PBS/Tween/CaCl buffer followedwith addition of a 1:20,000 dilution of Avidix-AP. Plates were furtherincubated for 1 hour at room temp and then washed 3× with PBS/Tween/CaClbuffer and 1× with Tris/MgCl/CaCl pH 9.8 buffer. 100 ul CSPD/Sapphire IIwas added and wells incubated for 30 minutes to room temp. Luminescencewas then measured in a TR717 microplate luminometer (Tropix).

[0023]FIG. 13. Inhibition of 12.5 nM rec HIV-1 Protease withAcetyl-Pepstatin. Assays consisted of reactions with 100 nM HIV FLAGPeptide in wells coated with Anti-FLAG MI antibody (0.1 ug/ml) for 1hour at 37° C. Dilutions of acetyl-pepstatin were preincubated for 5minutes with rec HIV-1 enzyme before adding to HIV FLAG peptidesubstrate in protease buffer. All additions were made on a Zymark RapidPlate. The plate was then washed 4× with TBS/3 mM CaCl₂/0.05% Tween onTecan 96 plate washer. 100ul of a 1:20,000 dilution of Avidix-APconjugate was added to each well and incubated for 1 hour @ room temp.The plate was then washed 3× with above buffer and 1× withTris/MgCl/CaCl pH 9.8. 100 ul CSPD/Sapphire II was added, incubated for30 min and then light emission was measured in a TR717 luminometer(Tropix). Note: Acetyl-pepstatin is a well known aspartic proteinaseinhibitor and is a reported inhibitor of HIV-1 protease (Pro. Natl. Sci.U.S.A. 85, 66123, (1988)).

[0024]FIG. 14. Synthetic peptide for homogenous assay. This peptide,also adapted for HIV-1 protease inhibitor detection, is derivatized atboth ends of the molecule, in fashion similar to that employed in theheterogenous assay. In this case, however, one terminus, preferably thecarboxy terminus, is labeled with an energy accepting fluorescentmoiety, such as fluorescein, and the amino terminus is labeled with a1,2-dioxetane moiety precursor (14A) which can be photooxygenated insitu to the dioxetane (14B). The dioxetane may be triggered by otherthan chemical (enzymatic or non-enzymatic) means if necessary.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] A peptide substrate is synthesized which contains the appropriatecleavage site for the target protease. This peptide is labeled, in aheterogenous assay, with one member of a first ligand binding pair, suchas biotin, on one end and a member of a second ligand binding pair, suchas fluorescein, at the other end. This peptide is then incubated withthe protease and a compound of interest to be screened for inhibitoryactivity, in a well or other solid phase coated with the second bindingligand of said first ligand binding pair, such as avidin or strepavidin.

[0026] The spacer between the ligand binding pair members and thepeptide of the inventive substrate may be a covalent bond or anycovalent binding moiety which does not interfere with either the firstor second ligand binding pair or substrate cleavage. Among exemplarysequences are C1-12 alkyls, alkylamino's, carboxylic acids, or anyneutral moiety terminating in coupling functionalities at either end.The spacer may be provided with water-solubility enhancing substituents(e.g., carboxy, sulfoxy, halo, etc.) or, where necessary forenzyme/antibody presentation, to cyclize the substrate, bridging agents.

[0027] After incubation, the wells are washed, incubated with the secondbinding member of said second binding ligand pair conjugated with anenzyme which is an effective trigger for a 1,2-dioxetane, such asalkaline phosphatase, washed, incubated with a 1,2-dioxetane substratesuch as chlorine substituted phosphate dioxetane (CSPD) and the signalis measured. Higher signals are detected in the presence of aninhibitor.

[0028] The 1,2-dioxetanes used as substrates may be any of thosedescribed in any of Tropix' prior patents, including U.S. Pat. Nos.4,931,223; 4,931,569; 4,952,707; 4,956,477; 4,978.614; 5,032,381;5,112,960, 5,154,772; 5,220,005; 5,225,584; 5,326,882; 5,330,900;5,336,596; 5,543,295; 5,582,980; 5,605.795; 5,625,007; 5,654,154; and5,679,803, which are incorporated by reference herein. The abovementioned patents disclose 1,2-dioxetanes as chemiluminescent compoundswhich can be used as reporter molecules in ultrasensitive assays thatcan be conducted quickly without resort to exotic conditions orelaborate apparatus, for the detection of a variety of biologicalmolecules. A preferred substrate is CSPD, the chlorine-substitutedcounterpart of AMPPD, which is the disodium salt of3-(4-methoxy-spiro[1,2-dioxetane-3,2′-tricyclo[3.3.1.1^(3.7)]decan]4-yl)phenylphosphate.

[0029] The dioxetanes contain a fluorescent chromophore group,preferably an aryl group such as phenyl or naphthyl and an enzymecleavable group, e.g., a phosphate ester, which when cleaved by theappropriate enzyme, e.g., alkaline phosphatase, forms a negativelycharged substituent (e.g., an oxyanion). This destabilizes thedioxetane, thereby causing the dioxetane to decompose to form twocarbonyl-containing groups accompanied by the release of light.

[0030] To enhance the chemiluminescent signal, and improve signal/noiseratio to permit discrimination between background signals and positivetarget-responsive signals at very low levels, a water-solubleenhancement agent may be added to the sample prior to or concomitantwith the introduction of the dioxetane. Specific enhancement agentswhich may be used include: quaternary onium polymeric salts such aspolyvinylbenzyltributyl-ammonium chloride (Sapphire II, Tropix) or anyof those disclosed in U.S. Pat. No. 5,336,596 as potential membranecoatings, neutral detergents such as Tween-20 (Sigma), cationicdetergents, such as cetyltrimethylammonium chloride (CTAB, Sigma) andcombinations thereof.

[0031] Families of proteolytic enzymes which may be targets fordetermination of inhibitory agents include

[0032] Cysteine Proteases

[0033] Caspases 1, 2, 3, 6, 7, 8

[0034] Cathepsins (B, H, S and L)

[0035] Hydrolase

[0036] L-proteinases

[0037] Calpain

[0038] Interleukin converting proteases (ICE)

[0039] Serine Proteases

[0040] Urokinase

[0041] Trypsin

[0042] Thrombin

[0043] Cathepsin G

[0044] Aspartic Proteases

[0045] HIV-1 and 2

[0046] Yapsin I and YAP 3

[0047] Plasmepsin I and II

[0048] Cathepsin D and E

[0049] Metalloproteinases

[0050] Collagenase

[0051] Gelatinase A and B

[0052] Stromelysin

[0053] Aminopeptidase

[0054] Elastase

[0055] or any of those mentioned in U.S. Pat. No. 5,591,591. Assayconditions and applications for specific proteases are also listed inU.S. Pat. No. 5,591,591. It is possible to use any peptide which isrecognized by the protease of interest. Examples of specific peptidesand proteases are listed in U.S. Pat. No. 5,591,591. In particular, apeptide is prepared which features a cleavage site for which the targetprotease is specific. The resulting peptide is short, and easilyprepared using conventional synthetic technology.

[0056] A large variety of ligand binding pairs can be used for both thefirst and second ligand binding pair employed. Among preferred labelsfor either end of the peptide are: Biotin, Fluorescein (FAM), FLAG, HIStag (6 histidine amino acid sequence), and Digoxin (digoxigenin labeledpeptide). These are bound by the other member of the first pair,preferably bound or attached to a solid phase so as to remain throughwashing and addition of the other member of the second pair, added afteran opportunity for cleavage to occur, the other member being complexedwith alkaline phosphatase or other suitable enzyme as a trigger. Theprincipal restriction on the identity of the binding pairs is that thefirst binding pair be distinct from, and not interact with, the secondbinding pair.

[0057] In addition to alkaline phosphatase, other enzymes which may beused to cleave the enzymatically cleavable group from the dioxetaneinclude: acid phosphatases, esterases, decarboxylases, phospholipaseD,β-xylosidase, β-D-frucosidase, thioglucosidase, β-D-galactosidase,α-D-galactosidase, α-D-glucosidase, β-D-glucosidase, α-D-mannosidase,β-D-mannosidase, β-D-fructofuranoside, β-D-glucosiduronase, and trypsin.

[0058] In addition to being of particular interest as organic moietiesthat constitute diagnostic markers, protease enzymes are also ofconsiderable interest as enzyme labels. Examples of diagnostic proteasemarkers include cathepsin B (cancer), cathepsin G (emphysema, rheumatoidarthritis, inflammation), plasminogen activator (thrombosis, chronicinflammation, cancer) and urokinase (cancer). Assays for proteasedetection are therefore needed to monitor protein stability in variousbiological and commercial processes.

[0059] In an alternative embodiment of this assay a homogeneouschemiluminescent energy transfer assay is provided. In this approach,one end of the cleavage sequence peptide bears a 1,2-dioxetane ordioxetane precursor. Direct attachment of this moiety, followed byoxygenation to form the dioxetane, is enabled in U.S. patent applicationSer. No. 08/767,479, allowed and incorporated herein by reference. Theother end of this peptide bears an energy accepting fluorescent moleculesuch as fluorescein, or any of a variety of similar fluorescingmoieties, such as those disclosed in U.S. Pat. Nos. 5,004,565 and5,208,148 which are incorporated herein by reference. The peptide issufficiently short (no more than about 10 amino acid residues) such thatthe dioxetane is in close physical association with the fluorescentlabel. Upon triggering of the dioxetane, which can be effected byaddition of an enzyme, or pH alteration, or application of heat or othertriggers, the dioxetane decomposes, emitting energy which excites thefluorescent moiety which then fluoresces if no cleavage has occurred (apositive test for protease inhibition). If cleavage has occurred, thedioxetane and fluorescent moieties are no longer in close physicalrelationship, and the light is emitted by the chemiluminescentdioxetane. The wavelength of the fluorescent emitter ischaracteristically shifted markedly from that of the dioxetane, allowingeasy discrimination in a homogenous assay.

[0060] Another approach to a homogeneous assay using an enol lether ordioxetane label is to use a quenching group in place of the fluorescentacceptor. In this format, upon triggering of the dioxetane label, therewill be no light from the intact peptide due to the quenching group. Ifcleavage of the peptide substrate by a protease occurs prior totriggering, emission from the dioxetane group will be observed upontriggering. This quenching process is subject to the same distancerestraints as the energy transfer process described above. A suitablequenching group for the substrate shown in FIG. 14 would be a dabcylgroup in place of the fluorescein group. Any non-fluorescent lightabsorbing dye, which has an absorption spectra which overlaps theemission spectra of the donor dioxetane could be used as a quenchinggroup.

[0061] Variations on this approach can be made for many types ofproteases. Direct covalent or hydrophobic attachment of a peptidesubstrate to a microwell surface may be required for some proteases.Alternatively, direct synthesis of the peptide on the solid phase mayalso have advantages.

[0062] The heterogenous assay of this invention has been exemplified,below, by reference to an assay adapted to detect inhibition of HIV-1protease. This example is demonstrative only, and not intended to belimiting.

[0063] Materials and Methods

[0064] Reagents:

[0065] 1. The HIV FLAG peptide was custom synthesized by GenemedSynthesis, Inc.

[0066] 2. Anti-FLAG MI Monoclonal Antibody, FLAG Octapeptide, andEnterokinase were purchased from Eastman Kodak Company.

[0067] Neutravidin coated plates: Neutravidin™ Biotin Binding Protein(PIERCE) was diluted to 5 ug/ml or 0.1 ug/ml in BupH™Carbonate-Bicarbonate buffer (PIERCE), 1 mg/ml BSA (fraction V Sigma)and 100 ul added to white 96 well plates (Dynatech-Microlitel) andincubated for 2 hours at 37° C. The wells were then washed with PBS,0.1% (v/v) Tween 20 and blocked with PBS, 0.1% (V/V) Tween 20, 1 mg/mlBSA overnight at 4 degrees.

[0068] Protease assays: Rec HIV-PR1 (affinity purified -BACHEM) wasdiluted in 0.1 M NaAcetate, 1M NaCl, 1 mM EDTA, 1 mM DTT, 1 mg/ml BSA,pH 4.7 or pH 5.5 and added to Neutravidin coated plates. Rec HIV-1inhibitor (Acetyl-pepstatin(BACHEM)), compounds from plate-1001 (SigmaDrug Standards) and/or DMSO was added followed with a 5 minutepreincubation before the addition of HIV-l peptides I or II,(FAM-spacer-Ser-Gln-Asn-Tyr-Pro-lle-Val-Gln-spacer-Biotin (Perkin-Elmer)or (FLAG-Ser-Nle-Ala-Glu-Phe-Leu-Val-Arg-Ala-Lys-His-Spacer-Biotin) orDABCYL-y-Abu-Ser-Gln-Asn-Tyr-Pro-lle-Val-Gln-EDANS (BACHEM) (prior art)respectively. Reactions were done at ambient and/or 37° C. For HPLCanalysis, reactions were stopped with 0.1% TFA and placed on ice beforeinjection. Assays with the BACHEM fluorogenic substrate were measureddirectly on a LS50B fluorescence spectrophotometer (Perkin-Elmer).Neutravidin coated assay plates with captured biotinylated-fluoresceinlabeled peptide I were washed with PBS, 0.1% (V/V) Tween20, 1 mg/ml BSAand incubated for 60 min. with anti-fluorescein-alkaline phosphatase Fabfragments (Boehringer Mannheim) at ambient temperature. Plates werewashed again with PBS buffer followed by a lOmM Tris-HCL, 1 mM MgCl₂ pH9.8 wash. 100 ul of CSPD/Sapphire II™ ready to use alkaline phosphatasewas added and emitted light was measured in a TR717® microplateluminometer(Tropix).

[0069] HPLC analysis: Analytical HPLC on the HIV-1 protease substratecleavage products was done on a Perkin-Elmer UV/Vis detector with series2001c pump, Perkin-Elmer pecosphere 5 C18, Sum, 4.6 mm×15 cm, gradientof water (0.1% TFA) and CH₃CN (0.1% TFA) from 30% to 60% over 10 minutesat 1 ml/min. Additions and dilutions were made with a ZymarkRapidPlate-96 pipeting station (Zymark Corp.) and washes were done witha Tecan 96PW washer (Tecan).

[0070] The present inventors have successfully developed an automatedscreening assay as shown below to search for novel HIV-1 proteaseinhibitors. AUTOMATION STEPS VOLUME TIME # DESCRIPTION (μL) AUTOMATIONCOMPONENT (MIN) 1 Add protease buffer 50 μL Zymark Rapid plate 96-wellpipettor 1 min containing 50 nM HIV-1 protease 2 Add test compound 5 μLZymark Rapid plate 96-well pipettor 1 min 3 Add HIV Perkin Elmer 45 μLZymark Rapid plate 96-well pipettor 1 min peptide @ 444 nM 4 Incubate(32° C.) 60 min 5 Wash 3 × 100 μL Tecan 96PW washer 0.75 min 6 AddAnti-Fluorescein- 100 μL Zymark Rapid plate 96-well pipettor 1 min APconjugate 7 Incubate (Room Temp.) 30-60 min 8 Wash 5 × 100 μL Tecan 96PWwasher 1 min 9 Add CSPD + Sapphire 100 μL Zymark Rapid plate 96-wellpipettor 1 min II 10  Read plate Tropix TR717 Luminometer 30 min + 1.4min

[0071] We have shown that this protease assay can be set up as anendpoint capture assay with minimal reagent use. An HIV-1 peptidesubstrate I concentration of 200 nM and a rec HIV-1 proteaseconcentration of 25-50 nM have been shown to be optimal workingconcentrations with good sensitivity and acceptable signal:noise. Aresonance energy transfer assay reported by Abbott Laboratories,Matayoshi. E. D. et al., Science 247: 954-958 (1990), utilizesmicromolar amounts of HIV-1 peptide II. This assay is reported as anassay with a linear detection window of less than 5 minutes, which wehave found to have a poor signal to noise ratio making it a difficultrobotic assay to automate. FIG. 6. This assay gave a maximum sin ratioof 4, while the inventive assay gives a sin ratio of greater than 140when using the FLAG/anti-FLAG ligand binding pair. By utilizing commonlaboratory reagents such as biotin and fluorescein one can eliminate theneed to generate sequence specific monoclonal antibodies as described inthe alternative HIV peptide capture based assay by Fournout, S. et al.,Anal. Chem. 69, 1746-1752 (1997). We have shown that DMSO is welltolerated in the assay. The IC₅₀ of acetyl-pepstatin in our captureassay was 2-3 uM compared to 0.3 uM reported by Fournout. S. et al.

[0072] This invention has been disclosed in terms of both genericdescription and specific example. Variations will occur to those ofordinary skill in the art, including peptide moiety identities, specificproteolytic enzymes to be employed, enhancement agents and enhancementadditives, and specific assay formats without the exercise of inventivefaculty. Such variations remain within the scope of the invention, savefor variations excluded by the recitation of the claims presented below.

1. A method for conducting an assay to determine whether a targetcompound exhibits activity as a protease inhibitor, comprising:combining said protease and said target compound in an environmentfurther comprising a construct, wherein said environment is such thatsaid protease cleaves said construct in the absence of inhibition ofsaid protease, said construct comprising an amino acid sequenceterminating, at a first end, with a first member of a first ligandbinding pair and at a second end with a first member of a second ligandbinding pair, wherein said first member of said first ligand bindingpair binds to a second member of said first ligand binding pair whichsecond member is bound to a surface, removing any fragments of saidconstruct cleaved by said protease, adding a complex of a second memberof said second ligand binding pair complexed with an enzyme and allowingsaid complex to bind to any said first member of said second ligandbinding pair present, removing any unbound complex, adding a1,2-dioxetane which is a substrate for said enzyme and observing anychemiluminescence released thereby, wherein emission of saidchemiluminescence is indicative of protease inhibition activity by saidtarget compound.
 2. The method of claim 1 , wherein saidchemiluminescence observed is measured, and wherein the amount ofchemiluminescence observed is correlated with the degree of inhibitoryactivity exhibited by said compound.
 3. The method of claim 1 . whereinsaid assay is a transfer free single plate endpoint assay.
 4. The methodof claim 1 , wherein said surface is at least a portion of at least onewell of a test plate.
 5. The method of claim 1 , wherein said secondmember of said second ligand binding pair is an antibody.
 6. The methodof claim 1 , wherein said first ligand binding pair is biotin and acompound which binds to biotin at least as tightly as avidin.
 7. Themethod of claim 1 , wherein said second ligand binding pair is biotinand a compound which binds to biotin at least as tightly as avidin. 8.The method of claim 6 , wherein said compound which binds to biotin atleast as tightly as avidin is avidin or strepavidin.
 9. The method ofclaim 7 , wherein said compound which binds as tightly as avidin isavidin or strepavidin.
 10. The method of claim 1 , wherein said firstmember of said first ligand binding pair is biotin and said first memberof said second ligand binding pair is fluorescein.
 11. The method ofclaim 1 , wherein said second ligand binding pair is FLAG and anantibody therefor.
 12. The method of claim 1 , wherein said protease isselected from the group consisting of a serine protease, a cysteineprotease, an aspartic protease and a metallo proteinase.
 13. The methodof claim 1 , wherein said protease is selected from the group consistingof HIV-1 protease, caspases, cathhepsins, hydrolase, L-proteinase,calpain, interleukin converting proteases, urokinase, trypsin, thrombin,HIV-2 protease, Yapsin I, Yapsin 3, Plasmepsin I, Plasmepsin II,collagenase, gelatinases, stromelysin, amino peptidase and elastase. 14.The method of claim 13 , wherein said protease is HIV-1 protease.
 15. Aprotease substrate for conducting an assay to determine proteaseinhibitory activity in a target compound, said substrate comprising: apolypeptide, said polypeptide including a cleavage site specific forsaid protease, said polypeptide bearing at a first end a first member ofa first ligand binding pair and at another end a first member of asecond ligand binding pair, wherein said first member of said firstligand binding pair and said first member of said second ligand bindingpair do not bind to each other.
 16. The substrate of claim 15 , whereinsaid first and second ligand binding pair is comprised, independently,of an antigen and an antibody therefore, or biotin and a compound whichbinds as tightly to biotin as avidin.
 17. The substrate of claim 15 ,wherein said substrate has the sequenceFluorescein-Spacer-Ser-Glu-Asu-Tyr-Pro-Ile-Val-Glu-Spacer-Biotin, orFLAG-Ser-Nle-Ala-Glu-Phe-Leu-Val-Arg-Ala-Hys-His-Spacer-Biotin.
 18. Ahomogenous assay for detection of protease inhibitory activity in atarget compound, comprising: combining said protease and said targetcompound in an environment further comprising a construct, wherein saidenvironment is such that said protease cleaves said construct in theabsence of inhibition of said protease, said construct comprising apolypeptide of 2-10 amino acids, said polypeptide terminating, at afirst end, and a moiety which is a 1,2-dioxetane or precursor thereofwhich can be oxygenated in said environment to provide a 1,2-dioxetanemoiety and, at a second end, with an energy accepting fluorescentmoiety, oxygenating said precursor if present to form a 1,2-dioxetanemoiety, causing said 1,2-dioxetane moiety to decompose, and observingthe wavelength of light emitted from said environment, wherein saidwavelength, if the wavelength of said fluorescent emitter, is indicativeof inhibitory activity, and said wavelength, if the wavelength of saiddioxetane, is indicative of absence of inhibitory activity.
 19. Aconstruct for conducting a homogenous assay to determine proteaseinhibitory activity in a target compound, comprising: a polypeptide of2-10 amino acid residues, wherein said polypeptide includes a cleavagesite specific for said protease, said polypeptide bearing, at one end, afluorescent energy accepting moiety or a chemiluminescece quenchingmoiety and at another end, a 1,2-dioxetane or 1,2-dioxetane precursormoiety that can be oxygenated to form a 1,2-dioxetane moiety in thecourse of said assay.
 20. A kit for conducting an assay to determinewhether a protease inhibitor is present in a sample, comprising in oneor more containers: (a) a peptide which is a substrate for saidprotease, wherein said peptide substrate is labeled with (i) a firstmember of a first ligand binding pair, and (ii) a first member of asecond ligand binding pair; (b) a chemiluminescent 1,2-dioxetanesubstrate containing an enzymatically cleavable group, which substrateis capable of producing light in the presence of an enzyme which cleavessaid enzymatically cleavable group from said substrate.
 21. The kit ofclaim 20 , wherein said protease is HIV-1.
 22. The kit of claim 21 ,wherein said peptide substrate comprises the amino acid sequence:-Ser-Gln-Asn-Try-Pro-Ile-Val-Gln-.
 23. The kit of claim 20 , whereinsaid first member of said second ligand binding pair is fluorescein. 24.The kit of claim 20 , wherein said first member of said first ligandbinding pair is biotin.
 25. The kit of claim 20 , additionallycomprising a polymeric quaternary onium salt enhancement agent for said1,2-dioxetane.
 26. The kit of claim 25 , wherein said enhancement agentcomprises polyvinylbenzyltributyl ammonium chloride.
 27. The kit ofclaim 20 , further comprising a enzyme complexed with a second member ofsaid second ligand binding pair.
 28. The kit of claim 27 , wherein saidenzyme is alkaline phosphatase.